A crude membrane system has been prepared that catalyzed the transfer of the oligosaccharide unit of (alpha-Man) 4-6-beta-Man-beta-GlcNAc-PP-dolichol to an ASN residue in certain proteins of known amino acid sequence. Preliminary results indicate that the protein must be unfolded before it will serve as an acceptor of the oligosaccharide chain and it must contain an ASN residue as part of the tripeptide sequence -ASN-X-SER- or -ASN-X-THR-, where X represents one of the 20 amino acids. However, the latter condition is not sufficient for a protein to serve as an acceptor, since a number of proteins possessing the tripeptide sequence do not serve as substrates for the oligosaccharide transferase. The objective of the proposed study is to gain an understanding at the molecular level of the factors that determine if a given protein will be glycosylated at a specific ASN residue in its primary structure. Oligosaccharide transferase will be purified from cell-free preparations from hen oviduct. Using the purified enzyme and two proteins (RNase A and alpha-lactalbumin) of known sequence that, after unfolding, serve as acceptors of the oligosaccharide, a systematic study of the structural requirements of the substrates will be undertaken. Peptide fragments of varying size containing the sequence -ASN-X-SER(THR)- will be isolated from RNase A and from alpha-lactalbumin, and tested as substrates for the oligosaccharide transferase. These studies, in conjunction with parallel studies on peptide fragments of proteins that contain the sequence -ASN-X-SER(THR)-, but are inactive as acceptors, should define the structure features required for a protein to serve as an acceptor. Further information on the factors determining the specificity of enzymatic glycosylation of proteins will be obtained by a comparative study of the activity of purified oligosaccharide transferases isolated from hen oviduct and from bovine thyroid toward proteins and polypeptide fragments of known amino acid sequence.